FUNDAMENTALS 2 Scribe: LOUISA WARREN

FUNDAMENTALS 2 Scribe: LOUISA WARREN 10-18-2010 Proof: CHRISTINE SIRNA YOTHER BACTERIAL PHYSIOLOGY Page 1 of 10

I. GRAM-NEGATIVES [S30]: a. Cytoplasmic membrane similar to that of gram positive b. Cell wall following that c. Then it starts to get different- outside of the cell wall, there is another membrane i. This is called the outer membrane (OM) ii. It is not like the cytoplasmic membrane (inner membrane) iii. OM is lipopolysaccharide (LPS), and the other structures on here are various proteins II. GRAM-NEGATIVES [S31] a. Gram negative differs from gram positive in that it has a very thin peptidoglycan (PG) layer- about 2 nm in structure i. If you were to take all of this PG and add it up, it is about enough to go around the cell one time ii. But it still has cross-links to give it integrity 1. Like the cross-links in the gram positive cell wall 2. Gives it a double layer with thickness of 2 nm 3. Still thin compared to gram positive cell wall b. No Teichoic acids in gram negative- they are unique to gram positive c. In between Inner and outer membrane is the periplasmic space i. Site of many digestive and protective enzymes ii. Some mechanisms that allow for antibiotic resistance contained here iii. Where transport occurs from the outside of the outermembrane to the inside of the cell iv. Where the PG is located d. Outer membrane is found outside the PG i. Primary function of OM: blocks entry of very large molecules (ones greater than 800 daltons) ii. PG is attached to the OM by a lipoprotein iii. The OM is not a typical lipid bilayer- it is made of an LPS, which is the outer leaflet of the OM iv. If you look back (S27), the inner leaflet of the OM has typical lipids (the black structure) and outside of that is LPS (green) 1. The outer leaflet is not typical in the bilayer v. Transport is important for the OM- things have to get through there to get to the cell, and there are specific mechanisms for that III. LIPOPOLYSACCHARIDE (LPS) [S32] a. LPS is endotoxin- responsible for toxic shock- fever, hypotension, all of the properties of toxic shock b. Looking at the membrane you see the inner leaflet, then the outer leaflet of the OM which is made of Lipid A, followed by the core polysaccharide further outside, then outside is the O Antigen (O Ag) c. Lipid A i. Anchoring component that allows LPS to be the outer part of the OM ii. Has the toxic properties of LPS (endotoxic shock) d. Core polysaccharide is short, made of 7 or so sugars i. Structure varies with different bacterial species ii. When you talk about the species, back to the Borrelia example 1. Borrelia was the genus 2. Burgdorferi was the species (the more specific part of the bacteria) 3. So the core polysaccharide will vary with the different Borrelia species, or the different Salmonella species, for example e. O Antigen i. Made of polysaccharide, repeating units that are 3-5 sugars in length to make a very long chain ii. Outermost surface of the cell is the O Ag polysaccharide iii. Can vary with particular strain 1. For example, within Borrelia Burgdorferi, there can be strain A, B, C, or D- different isolates 2. So the O Ag can vary with different isolates of the same bacterial species 3. Becomes important in serotyping a. For example, E. Coli has hundreds of O Ag b. Can be serotyped using antiserum specific for individual O Ag c. E. Coli O157- O refers to the O Antigen, and that is the serum type IV. OPTIONAL FEATURES (GRAM +/-) [S33] a. Remember: i. Unique to gram positive- teichoic acids, lipoteichoic acids, thick cell walls ii. Gram negative- thin cell walls, OM, LPS FUNDAMENTALS 2 Scribe: LOUISA WARREN 10-18-2010 Proof: CHRISTINE SIRNA YOTHER BACTERIAL PHYSIOLOGY Page 2 of 10 b. These features can occur on either gram positive or gram negative cells c. These features may not be essential for viability, but may make it possible for the cells to be pathogenic organisms that cause disease or survive in multiple different environments d. Capsules i. Almost always made of polysaccharides 1. A few exceptions 2. Bacteria that causes anthrax has capsule made of protein ii. Can work in a couple of different ways to block the complement or antiphagocytosis 1. The cell wall, particularly in gram positive, activates complement with C3b binding of the complement 2. Phagocytic receptors normally recognize this and allow it to be phagocytized 3. Capsule can come along and block the phagocytic receptors from being able to recognize that deposited complement 4. Alternatively, capsule can be there to block activation of complement 5. So it can either block activation or block the recognition if activation has occurred and C3b has deposited 6. In the gram negative cell, the O Ag would be the outside part, and what activates the complement is the LPS a. The blocking can occur in the same way- either preventing it from being recognized or from ever taking place e. Surface Proteins are anchored i. Can be anchored in the teichoic acid, PG, OM ii. Also serve functions of attachment and antiphagocytosis (both capsules and surface proteins can serve attachment functions) iii. One important polysaccharide that serves an attachment function is an oral bacteria, streptococcus mutans 1. Has an outer polysaccharide glucan polymer that is essential for being able to attach to the surface of the tooth 2. In the absence of that, it is not able to anchor and cause dental caries iv. One of the bacteria that causes disease- streptococcus pneumoniae- causes pneumonia in the elderly, recurrent ear infections in children, also causes many eye infections 1. The capsule is an absolutely essential component for it to cause systemic disease 2. Non-encapsulated organisms are what cause eye infections 3. So it is a unique part of the environment, and eye infections are the only instance where capsule is not essential for streptococcus pneumoniae f. Flagella are native proteins i. Structure shown in color ii. Have a basal hook/body that anchors them to membranes iii. Flagella spins around- this allows for motion to propel the cell iv. For motility, chemotaxis, getting bacteria to things that are good and away from things that are bad, and important in virulence v. Many different kinds 1. Peritrichous- all around the cell 2. Unipolar- on one end of the cell 3. Bipolar- two g. Capsules in pictures on left show when bacteria is grown on agar plates i. Strain on the right is the same as what is on the left ii. Right group is unable to produce capsule- so what you see in the colony on a plate (left) is the large amount of capsule the bacteria is able to make iii. Under the microscope, the chains with black dots are streptococci growing in the cell 1. This is a capsule that has been reacted with an antibody to emphasize the capsule, and you can see large amounts of capsule around the cell h. Bacteria that make capsules have a huge amount on the outer surface of the cell V. OPTIONAL FEATURES (GRAM +/-) [S34] a. Pili are proteins, shorter and smaller than flagella i. Many involved in attachment ii. Others involved in gene transfer (in later lecture) b. Toxins i. Excreted from the cell to act on host cells ii. Not the same as endotoxin 1. Endotoxin is LPS, which is uniquely gram negative FUNDAMENTALS 2 Scribe: LOUISA WARREN 10-18-2010 Proof: CHRISTINE SIRNA YOTHER BACTERIAL PHYSIOLOGY Page 3 of 10 2. Endotoxin is not excreted from the cell; it remains attached with the cell iii. Toxins can be made by gram positive or gram negative bacteria, and they act on host cells c. There are many enzymes that bacteria make i. Hyaluronidases, proteases, DNAses ii. Need to be able to degrade things in their environment not only to survive by utilizing them as nutrition but also to invade through tissues d. Endospores are dehydrated cells i. Survive long-term in dry conditions and extreme environments- why they are useful in terrorist activities ii. Can rehydrate to form normal bacterial cell, which can cause disease 1. Anthrax spores can be inhaled and germinate inside the body/ lungs iii. Mainly formed by 2 bacteria 1. Clostridium- causes food poisoning/ botulism- spores are important in passing the disease along 2. Bacillus VI. BACTERIAL GROWTH AND METABOLISM [S35] VII. GROWTH REQUIREMENTS [S36] a. Bacterial cell is mostly made of water- about 70% is water, and the rest is what we talked about in the beginning of lecture b. Carbon and energy sources are required for growth for all bacteria i. These may be the same thing ii. Most bacteria and all of the ones that are pathogens are chemoheterotrophs- use an organic molecule for both their carbon and energy source 1. Some of the things they can use frequently are glucose, galactose, all kinds of monosaccharides, disaccharides, organic acids, amino acids, alcohols, ribitols, fatty acids 2. There is probably some bacterium somewhere that can grow on anything c. A lot of these are utilized in the laboratory not only to grow bacteria but to help identify bacteria based on what conditions they will grow under VIII.GROWTH REQUIREMENTS- NITROGEN [S37] a. Nitrogen is a requirement and can come from 2 ways: inorganic source or organic b. Inorganic i. Many bacteria can take ammonia and ultimately convert that to Glu or Gln ii. Others can fix nitrogen to ammonia and then to Glu/Gln iii. Some are able to do nitrate reduction to get ammonia iv. Others are denitrifiers- a different thing/ utilization v. All ultimately get this into pathways as Glu or Gln c. Organic source i. Some bacteria are able to take up Glu and Gln directly and utilize them in pathways IX. GROWTH REQUIREMENTS- OXYGEN [S38] a. Important for bacterial cell and whether it is able to grow in oxygen for its own survival, but also important clinically (as are all growth requirements) because it is important to process samples in an appropriate way so that they are able to make it to the laboratory where they can be identified i. Oxygen is important because some cannot survive in aerated environments, and the reason goes back to their basic metabolism b. There are 5 ways to divide bacteria regarding their oxygen requirements. These are the 2 extremes: i. Strict Aerobe- requires oxygen under all circumstances to be able grow 1. Cannot ferment (ferment- transfer electrons and protons directly to an organic receptor) 2. Always have to respire (transfer to oxygen) to grow ii. Strict Anaerobes- Killed in O2 1. The ones you have to worry about transporting, they may not survive for too long in oxygen 2. Always ferment/ transfer to an organic receptor 3. Lack the enzymes necessary to degrade toxic oxygen metabolites iii. Oxygen itself is not toxic- biproducts of oxygen metabolism are toxic 1. Oxygen can be converted to hydrogen peroxide a. Toxic to many bacteria b. Avoid that toxicity through an enzyme called catalase, which breaks this back down to water and oxygen 2. Oxygen can also be converted to a superoxide radical a. Toxic b. The enzyme superoxide dismutase breaks that down to oxygen and hydrogen peroxide, which can then be converted by catalase to oxygen and water FUNDAMENTALS 2 Scribe: LOUISA WARREN 10-18-2010 Proof: CHRISTINE SIRNA YOTHER BACTERIAL PHYSIOLOGY Page 4 of 10 3. Anaerobes are strict anaerobes because they lack catalase and superoxide dismutase c. There are bacteria between aerobes and anaerobes i. Some lack these enzymes, some only have the machinery for respiration or fermentation X. GROWTH REQUIREMENTS- OXYGEN [S39] a. Facultative organisms are able to grow in the presence or absence of oxygen- they do whatever the environment dictates i. In oxygen, they respire ii. In the absence of oxygen, they ferment b. Aerotolerant anaerobes are able to grow with or without oxygen i. Don’t have machinery to respire ii. Always do fermentation iii. Tolerate oxygen because they have the enzymes to degrade the toxic biproducts, but they don’t utilize oxygen in their growth c. Microaerophiles grow best with low oxygen conditions i. Able to grow without oxygen but prefer to have oxygen XI. GROWTH REQUIREMENTS [S40] a. Temperature i. Most pathogenic organisms are mesophiles- optimum growth temperature at 20-40 degrees C ii. There are bacteria that can grow in extremes 1. High temperatures above 50 2. Low temperatures down to 4 and up to 20 degrees iii. Most pathogens prefer the mid-range- even though some of them can survive at lower temperatures, they will grow best at the higher temperatures b. pH i. Grow best at 6 to 8/mostly neutral range, but bacteria will have to survive extremes of pH 1. Bacteria that cause gastrointestinal diseases have to go through the stomach or intestines and be able to survive in the acid of the stomach if they are going to cause disease c. Many bacteria that cause disease are able to do so because they can survive in a given environment i. Bacteria that causes pneumonia doesn’t cause GI disease because it can’t survive in the GI tract ii. They have been selected for by the human host to cause the diseases that they cause- you are causing selective pressure on these with the different environments (pH, aeration, etc) d. Other things required for growth are usually considered trace elements, but they have to be there i. Sulfur, phosphorus, various minerals ii. Growth factors, etc. iii. The chromosome size is where this “other” can be very small or very large, depending on whether the bacterium is able to synthesize these things on its own 1. Many can synthesize all the amino acids they need, so you don’t have to add them 2. With others like the mycosplasma, you have to add serum and other things to get them to grow in culture XII. NUTRIENT UPTAKE [S41] a. Bacteria have to be able to take up the nutrients they are going to survive on, and one of the ways of doing that is by hydrolyzing those using various proteases, nucleases, lipases b. Have to be able to break down nutrients into something they can take in c. Once they are broken down, they have several protein-mediated pathways to take them in i. Facilitated diffusion ii. Active diffusion iii. Active transport iv. First three sides break this down and the last sums it up XIII.FACILITATED DIFFUSION [S42] a. Passive- doesn’t require energy to take place b. Carrier protein equilibrates the substrate in and out of the cell c. Once the substrate gets into the cell, it becomes phosphorylated i. Phosphorylated compounds do not pass in and out of the cell ii. If it is phosphorylated outside the cell, it can’t get in iii. So the cell brings it in and phosphorylates it on the inside so that it stays trapped on the inside d. Mechanism keeps going until it equilibrates the inside with the outside e. Glycerol is an example of a facilitated diffusion substrate XIV. ACTIVE TRANSPORT-GROUP TRANSLOCATION [S43] a. Requires energy: phosphoenolpyruvate or ATP FUNDAMENTALS 2 Scribe: LOUISA WARREN 10-18-2010 Proof: CHRISTINE SIRNA YOTHER BACTERIAL PHYSIOLOGY Page 5 of 10 b. Concentrates substrate in the cell rather than equilibration c. Substrate inside the cell is altered by phosphorylation to trap it in cell d. Glucose is an example XV. ACTIVE TRANSPORT-SUBSTRATE TRANSLOCATION [S44] a. Requires energy: proton gradient or ATP b. Carrier protein concentrates substrate in the cell c. In this case, substrate in the cell remains unchanged i. Does not get phosphorylated ii. This transport system recognizes the substrate on the outside of the cell to transport it in, but the carrier protein cannot recognize the substrate on the inside of the cell to take it back out 1. Has to do with the structure of the proteins involved XVI. PROTEIN-MEDIATED TRANSPORT MECHANISMS [S45] a. Is energy required? i. No for facilitated diffusion ii. Yes for active transport b. What happens to the substrate? i. Trapped by phosphorylation for facilitated diffusion and group translocation 1. Equilibrated in one and concentrated in the other ii. Substrate translocation is concentrated but unchanged c. Examples on the right side i. PTS- phosphotransfer system (the mechanism for doing this) XVII. BACTERIAL GROWTH IN CULTURE [S46] a. Bacteria are microscopic organisms- you have to look under the microscope to see them b. Can grow them in liquid or solid culture, but we don’t see individual bacteria without a microscope- you see the collective group c. In culture i. Usually done in a test tube or flask, under appropriate conditions (right media, right aeration, etc) ii. When you first inoculate a small amount of bacteria into the culture medium, it will be very clear, and it will grow for a long time before you start to see the turbidity change iii. It first starts to be barely visible at about 107 bacteria/mL iv. In the lab, the density will range from 108 to 1010/mL by the time they are turbid and ready for you to work with them 1. 108 /mL will be pretty turbid 2. 1010/mL will be saturated v. Realize that what is going on in culture is not necessarily what happens in the host/ in the real world 1. It may grow much more slowly there 2. How they grow depends on the environment vi. In a liquid culture, you measure optical density (OD) over time or colony forming units/mL 1. You see a lag phase early on a. There is not much of an increase in numbers b. Bacteria are getting ready to grow c. They are very actively metabolizing and getting larger (not just sitting there) vii. Then go into a log phase 1. Exponential growth 2. Divide very rapidly 3. Metabolize as quickly as they divide viii. At some point, they reach a stationary phase 1. Growth slows down or levels off 2. Two possible reasons: a. Slow growth because nutrients becoming limited b. Accumulating toxic products ix. Death phase 1. Also exponential 2. Caused naturally or induced by detergents, antibiotics, heat, or other causes x. Length of each phase depends on the type of bacteria 1. Some grow up to stationary phase and very quickly die 2. Some stay in the stationary phase for days or weeks before death 3. Depends on bacterium, what properties it has, what enzymes it has that direct it towards death or direct it to being able to remain viable FUNDAMENTALS 2 Scribe: LOUISA WARREN 10-18-2010 Proof: CHRISTINE SIRNA YOTHER BACTERIAL PHYSIOLOGY Page 6 of 10 4. Culture density you are able to achieve depends on the bacterium, and growth rate depends on bacterium a. E. Coli can double every 20 minutes under optimum lab conditions b. Others 1-2 hours, some even days for optimum doubling time c. E. Coli in suboptimal growth conditions can take an hour XVIII. BACTERIAL GROWTH IN SOLID/AGAR MEDIUM [S27] a. You can also culture bacteria in sold/agar medium i. What you put in the medium depends on the bacteria ii. Some will grow in blood agar plates b. This is E. Coli grown in a very minimal medium, which turns out to be very clear c. Some bacteria may require blood or serum in the plates, etc d. There are 100-200 colonies on this plate i. In order to get this, you take about 108/mL in the liquid and plate the liquid on here ii. To get this number of colonies, you have to dilute this several thousand-fold to plate it on here iii. Each one of these colonies on the plate arose from a single cell in the culture, so you have to dilute the 107 down to about 102/mL or so to be able to see individual colonies e. If the bacteria are streptococci, each one of these colonies would arise from a single chain of streptococci f. If we had staphylococci, each of these colonies would arise from a single cluster g. If you plate too many, you get a confluent growth and cannot see the isolated colonies i. You have to dilute the cells so that each cell can grow into a big colony h. How many cells do you think are in one of these colonies? i. Started with one cell (a microscopic organism) ii. Remember it is about 10^7 cells/mL to see any in liquid iii. 1015 would be concrete- some paper said they had that, but that would be paste iv. To start to see a colony on a plate would take about 107 v. This picture is probably about 108 cells in one colony vi. So it only takes a tiny little speck and you have billions of bacteria in there 1. The infectious dose for some bacteria can be on the order of 10 2. Others may take thousands, or 105, or 106, or 1010 3. 1010 is about a hundred of these colonies, but 108 is one of these dots (the head of a pen) vii. So there are a lot of bacteria in this small space XIX. BACTERIAL TAXONOMY [S48] a. How bacteria are named, classified, and identified b. Clinically important for determining what type of bacteria you are dealing with, what type of antibacterial is important to give under different circumstances c. This is all how these were identified and named and how we got where we are XX. BACTERIAL TAXONOMY [S49] a. Nomenclature- assigning names by international rules i. For example, Escherichia coli is always latinized and italicized ii. If you write it out on a piece of paper, you underline it iii. The first time you use it in a paper, you write out the full name (Escherichia coli) and after that you can use E. Coli b. Classification is the arrangement into taxonomic groups based on various similarities c. Identification is determining which group a new isolate belongs to d. The standard reference for this is Bergey’s Manual of Systematic Bacteriology which keeps everything up to date on how things are named etc. XXI. BACTERIAL NOMENCLATURE [S50] a. Did this slide before XXII. NUMERICAL CLASSIFICATION [S51] a. In the old days, bacteria were classified based on similarities and differences that were present- this is called numerical classification b. What they did was take bacteria, analyze them in all the ways we have talked about today, and make a big chart with +/- to find what each type most closely resembled i. Looked at everything under the miscroscope- size, shape, motility, does it make spores, is it gram positive or gram negative, does it stain, how does it grow on a plate, colony size, pigmentation ii. Biochemical/physiological traits- what are the conditions is best grows under? Temperature, sugar source, carbon source, oxygen requirements, etc c. This is how things were originally classified into groups- if you look back at some names, they have changed FUNDAMENTALS 2 Scribe: LOUISA WARREN 10-18-2010 Proof: CHRISTINE SIRNA YOTHER BACTERIAL PHYSIOLOGY Page 7 of 10 i. Streptococcus pneumoniae used to be called diplococcus pneumoniae because it wasn’t clear that it was a streptococcus until other things started to evolve and show that it was ii. Names of many other bacteria have changed over time because the classification and identification have gotten more specific, and we are better able to tell which bacteria belong with each other iii. When it is decided that a bacterium belongs more closely to one group than another, it can be slow to change the name because people are used to the old name, but they do change gradually with new information d. This is the original way of doing this. We will talk about some similar ways and then the specific way that is used now XXIII. SEROLOGICAL CLASSIFICATIONS [S52] a. E. Coli O157 is a serological classification i. These are all E. Coli, they all make O antigens, and that particular E. Coli makes O Ag number 157 b. Usually surface antigens- react with specific antibodies on different bacteria, usually surface i. We talked about the O Ag ii. Capsules 1. Streptococcus pneumoniaie a. Causes pneumonia and recurring ear infections in young children b. Has over 90 different capsular polysaccharides c. They will only express one at any given time, but it is important epidemiologically to know which ones are present- for example, with vaccines c. Important in epidemiology i. Vaccines are directed against specific antigens on the surface in streptococci pneumoniae (as in many bacteria) 1. In strep pneumo, where up to 90 capsular polysaccharides can be produced, it turns out that only strains making a small percentage of those are actually involved in disease 2. Serotypes may differ geographically a. In this country, a certain collection of serotypes are found, and in another country, another collection may be important b. It is important to know what are the serotypes that are causing disease in order to make the appropriate vaccine i. The vaccine that is now given to kids for the pneumococci is formulated from the 13 most common capsular serotypes in this country- that has changed over the past few years from 7 to 10 to 13 ii. The only way to know which serotypes to put into a vaccine is to know which serotypes are present in a given locale ii. These are not important for identification of bacteria- that is done in different ways, but they are important epidemiologically for knowing what exists in a given environment XXIV. GENETIC RELATEDNESS [S53] a. If you get to something that is more specific about how bacteria are related, you have to look at genetics b. There are various ways these are done- some are done because they are more convenient (for epidemiological studies), some are more accurate (for seeing if they are evolutionarily related) c. Genetic Relatedness focuses on looking at multilocus enzyme electrophoresis i. Looking at whether or not the specific enzymes are similar between two bacteria ii. The two bacteria may be altered by a few amino acid changes between the enzymes- these are enough to allow you to detect differences in the protein iii. How this works 1. You take a reference sample and unknown samples 2. Run the proteins out on a starch gel 3. There are various assays you can use to detect the positions of each of these migrations XXV. MULTILOCUS ENZYME ELECTROPHORESIS [S21] a. How this works i. You take a reference sample and unknown samples ii. Run the proteins out on a starch gel iii. There are various assays you can use to detect the positions of each of these migrations iv. Reference (on the right) is where the proteins will migrate, and you compare to that 1. So in this case (#2) the red enzyme is the same, but blue, green, and purple are altered at #2 2. In #1, they are all migrating the same b. These changes in migration are related to changes in amino acids in the protein, so you look at how many times it differs from the reference FUNDAMENTALS 2 Scribe: LOUISA WARREN 10-18-2010 Proof: CHRISTINE SIRNA YOTHER BACTERIAL PHYSIOLOGY Page 8 of 10 i. The more it differs, the more different it is from your reference organism ii. The more similar it is, the more closely related it is c. A lot of times you would use this with streptococcus pneumoniaie strains for example i. You would take a lot of different streptococcus pneumoniaie strains to look at how they vary in this regard ii. You might be able to tell how closely related they are iii. Gives you a lot more information than “their capsule is the same” or “their capsule is different”- you can get 10s or 100s of enzymes out of this kind of information XXVI. GENETIC RELATEDNESS [S55] a. We will skip ability to recombine and exchange DNA because it’s in the next lecture b. Bacteria that are more closely related are able to exchange DNA better than bacteria that are evolutionarily distant from each other- we will talk about that mechanism later XXVII. RFLP ANALYSIS [S23] a. DNA restriction profile is very similar to multilocus in the way it is interpreted i. Isolate DNA from your sample and a reference ii. Digest with a restriction enzyme iii. Run on an agarose gel iv. Stain with ethidium bromide to see the bands b. Again, you are looking for how many of these bands correspond to a reference sample- the more closely they correspond, the more similar they are c. Restriction enzymes cut at a specific site in the DNA, so the more closely related the DNA is, the more frequently those sites will occur at the same place in the DNA XXVIII. GENETIC RELATEDNESS [S57] a. DNA Base composition- the GC + AT content of any organism adds up to 100% i. If we just want to look at the GC content, we can refer to it as % GC, and you automatically know what % AT is b. There are many ways to do this by thermal experiments, etc with DNA to determine what % of the DNA are GC base pairs c. If the GC content for 2 different organisms is very different, you can conclude definitely that they are not related d. If GC content is very similar, you cannot conclude anything i. If 2 organisms are related, they must be similar ii. But just because they are similar doesn’t mean they are related XXIX. GC CONTENT [S58] a. Eubacteria have a very wide range of GC content b. Vertebrates have a very narrow range of GC content c. Some vertebrates can have a GC content of 40%, and many bacteria can have a GC content of 40%, but obviously those are very different organisms d. So just because it is the same, it doesn’t tell you anything- it tells you that they may be related if other things you find are also related e. If they are very different- if one is 40% and one is 60%- they will not be related organisms XXX. GENETIC RELATEDNESS [S59] a. DNA hybridization allows you to look at total or specific DNA sequences b. How it works i. Take DNA and spot it onto a matrix ii. Come back with labeled DNA from an unknown iii. The darker indicates more binding between it iv. The more binding, the more hybridization between the known and the unknown, the more closely related they are 1. If they have a lot of genes that are similar, they will bind 2. If a few, not so much XXXI. DNA HYBRIDIZATION [S60] a. This is exactly the same as a Southern Blot, except this is looking at the whole DNA instead of a specific sequence of DNA i. Southern Blot usually looks at one specific sequence XXXII. DNA HYBRIDIZATION- PCR [S61] a. PCR is a form of DNA hybridization where you take a primer specific for the DNA (in this case, specific for a given bacteria) b. If that primer is able to amplify and generate dsDNA, that sample is specific for the DNA you are looking at FUNDAMENTALS 2 Scribe: LOUISA WARREN 10-18-2010 Proof: CHRISTINE SIRNA YOTHER BACTERIAL PHYSIOLOGY Page 9 of 10 c. PCR is used frequently in laboratories to amplify a sequence, and what is used is a primer that is very specific for a specific gene in the bacterium of interest so that the only thing that gets amplified is from that bacterium XXXIII. GENETIC RELATEDNESS [S62] a. Skip DNA-RNA homology because similar to rRNA sequencing b. Ribosomal RNA sequencing- the most useful if you want to classify bacteria and determine evolutionary relationships c. Looking at DNA-RNA homology would be between specific DNAs and ribosomal RNAs that are highly conserved, but if we go one step further and do ribosomal RNA sequence, it becomes even more useful d. Ribosomal RNA sequencing is determining the sequence of the DNA that encodes rRNAs XXXIV. SENSITIVITY OF rRNA [S63] a. rRNA is associated with the ribosome and critical for protein synthesis b. DNA is transcribed to mRNA, then translated to protein c. In order to initiate synthesis, the rRNA has to bind to the initiation site/ ribosome binding site in the mRNA XXXV. TRANSLATION INITIATION [S64] a. This (yellow circle) is the ribosome b. Black letters show the 3’ end of the rRNA that is associated with the ribosome c. Blue is the mRNA that has been transcribed i. Within the mRNA of the bacteria is the Shine-Delgarno sequence and the initiation codon AUG ii. These together comprise the ribosome binding site iii. The Shine-Delgarno sequence is complementary to the 3’ end of the rRNA, and that is what allows the ribosome to recognize the mRNA 1. So this is a critical sequence in the rRNA 2. In different bacteria, the ribosome binding sites may vary a little, so this becomes relevant for recognizing these sequences XXXVI. SENSITIVITY OF rRNA [S65] a. rRNA is critical because it binds to the initiation site b. It also must have a very intricate secondary structure because it base pairs with itself, and that is very important for it to be able to bind XXXVII.SECONDARY STRUCTURE OF rRNA [S66] a. These loops are all bases that pair with each other i. Essential to the function of rRNA ii. If you had any mutations, these loops would not form b. So mutations that disrupt the structure not only disrupt the rRNA but also interfere with protein synthesis, and if you don’t have protein synthesis, you will die c. Structures are important, and small mutations in the structure could allow it to be disrupted XXXVIII. SENSITIVITY OF rRNA [S67] a. Changes in the critical areas are likely detrimental b. For that reason, DNA that encode rRNAs are highly conserved among bacteria (and all organisms) that are of common ancestry i. Their rRNAs will be very similar because of the critical nature of maintaining that secondary structure c. Sequencing the DNA of the rRNA of different organisms allows you to develop a phylogenetic tree- which ones are more closely related to others i. Phylogenetic trees are now based on rRNA sequences XXXIX. PHYLOGENETIC TREE [S68] a. The basic one for bacteria- these are the eubacteria b. Remember the tree from the first slides that showed eukaryotic organisms, archaebacteria, and eubacteria going in one direction- this is where that comes from c. Gram-positive bacteria form a tightly related group i. As a group, gram-positives are more closely related than gram-negatives because they are together on the tree ii. Everything else on here is gram negative- much more divergent d. Gram-positives can be divided into two large groups: i. Low GC species- have a low GC content ii. High GC species e. Mycobacteria and Mycoplasma don’t stain in the gram stain i. But in this type of characterization, Mycoplasmas fall into the low GC species gram-positive group ii. Mycobacteria fall into the high GC gram-positive group iii. When gram stains were originally done, it was not known what was going on FUNDAMENTALS 2 Scribe: LOUISA WARREN 10-18-2010 Proof: CHRISTINE SIRNA YOTHER BACTERIAL PHYSIOLOGY Page 10 of 10 iv. We ultimately figured out that it was a difference in cell wall and more than just that because gram-positives truly are a group together and gram-negatives spread out more but are clearly distinguished from the gram- positive even on an evolutionary basis f. These things get updated frequently because of more and more sequencing of this type [End 49:52 mins]